Human mesenchymal stem cells (hMSCs) are of great interest as a multipotent autologous cell source that have shown promise in the treatment of a variety of diseases, including osteogenesis imperfecta and left ventricular dysfunction. Better understanding of hMSC lineage commitment will allow the development of superior regenerative medicine therapies. Traditionally, hMSC differentiation has been carried out in vitro using a variety of soluble factors, which often fail to mimic physiologic conditions. The role of biomechanics in influencing hMSC differentiation has recently been investigated. Along with substrate stiffness, initial studies in which cells were cultured on protein patterns of arbitrary shape show that cell shape also has a strong effect on hMSC differentiation. Although a relationship between cell shape and differentiation has been noted, complete lineage-specific differentiation has not yet been achieved using cell shape as the primary effector. In this work, we will attempt to achieve more complete lineage-specific differentiation of hMSCs by directly regulating the cytoskeletal tension experienced by these cells. It is hypothesized that hMSCs that are restricted to making focal adhesions directly mimicking the adhesions of fully differentiated cells will preferentially differentiate to these specific lineages. This hypothesis ill be tested by analyzing the differentiation of hMSCs cultured on protein patterns that depict the adhesion sites of fully differentiated cells including osteoblasts, adipocytes, and neuronal cells. Laser scanning lithography will be used to develop these biologically inspired patterns by applying virtual masks of the adhesion sites of these differentiated cells to selectively desorb regions of an oligo(ethylene glycol) (OEG) terminated alkanethiol self-assembled monolayer; these patterened regions will be backfilled with human plasma fibronectin. This will yield adhesive protein patterns mimicking the adhesion sites of fully differentiated cells against a protein-resistant OEG background. Culturing hMSCs on arrays of these patterns and examining lineage-specific differentiation markers and gene expression will elucidate whether these patterns can direct hMSC differentiation to the same lineage from which they were derived. The effects of substrate rigidity in combination with adhesion site configuration will also be studied. The proposed research is broken down into the following specific aims: 1: Develop biologically inspired adhesive protein patterns on two-dimensional self-assembled monolayer surfaces. 2: Analyze hMSC behavior on micropatterned surfaces developed through specific aim 1. 3: Simultaneously test the impact of varying substrate mechanical properties and adhesion site configuration on hMSC differentiation. Successful completion of these aims will lead to an enhanced understanding of hMSC mechanobiology and has the potential to greatly impact the design of future stem cell therapies. PUBLIC HEALTH RELEVANCE: Although human mesenchymal stem cells (hMSCs) have shown great promise in regenerative medicine, a better understanding of the factors that influence hMSC differentiation is needed to improve future therapies. Materials based approaches that attempt to mimic biomechanical cues received by hMSCs in vivo have not been able to achieve complete lineage-specific hMSC differentiation in vitro. The proposed research will attempt to achieve more complete lineage-specific differentiation of hMSCs by culturing these cells on biologically inspired micropatterned surfaces, providing insight on stem cell mechanobiology that can greatly improve the design of stem cell therapies.